ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical function in T cell antigen receptor (TCR) signaling and in most aspects of T cell biology. ZAP-70 is an attractive therapeutic target for treatment of prevalent human T-cell dependent autoimmune diseases (e.g. rheumatoid arthritis, lupus, multiple sclerosis, psoriasis) and in allogenic transplantation. Considerable effort has previously been expended to develop specific ZAP-70 catalytic site inhibitors, without success, likely due to specific structural attributes of the kinase catalytic domain. Following TCR stimulation, the ZAP-70 tandem SH2 domains associate with dual tyrosine phosphorylated residues located within conserved sequence motifs (referred to as ITAMs) of the cytoplasmic domains of the CD3 and zeta chains of the TCR. Mutagenesis and structural studies on full-length ZAP-70 have shown that, prior to association with the ITAMs, ZAP-70 exists in an autoinhibited conformation in which the tandem SH2 domains stabilize an inactive conformation of the kinase domain. This proposal is built on the effort of the Kuriyan and Weiss labs in an NIAMS-funded 2009 ARRA Grand Opportunity grant to identify small molecules that stabilize the autoinhibited conformation of ZAP-70 and serve as allosteric inhibitors of the kinase. That successful research program led to development of a series of specific biochemical tools for identifying prototypical allosteric inhibitors of ZAP-70, and is poised to enable a dedicated new drug discovery effort in T cell regulation. In this current proposal, Nurix Inc., an emerging biopharmaceutical company founded by John Kuriyan, Art Weiss and Michael Rape, aims to extend this earlier work by optimizing the potency of allosteric inhibitors of ZAP-70 that could serve as a leads for clinical development of a new T-cell specific immunosuppressant. The program will (i) seek insight into the mode of interaction of these small molecule inhibitors with ZAP-70 using crystallographic and other biophysical techniques to facilitate lead optimization, and (ii) examine the activity of allosteric inhibitor compounds in established models of T cell function.